Advanced Clinical Imaging and Tissue-based Biomarkers of the Eye for Toxicology Studies in Minipigs

Abstract

There is increased interest to use minipigs in ocular toxicology studies due to their anatomical similarities with human eyes and as a substitute for nonhuman primates. This requires adaptation of enhanced optical coherence tomography (OCT) techniques and of ocular relevant immunohistochemistry (IHC) or in situ hybridization (ISH) markers to porcine eyes. In this study, OCT and OCT angiography (AngioOCT) were performed on adult Gö ttingen minipigs. To increase structural information on retinal and choroidal vasculature, OCT data were speckle denoized and choroidal blood vessels were segmented with threshold filtering. In addition, we established a set of IHC and ISH markers on Davidson's fixed paraffin-embedded minipig eyes: neurofilament-160, neuronal nuclei, calretinin, protein kinase C-a, vimentin, glial fibrillary acidic protein, glutamine synthetase, ionized calcium-binding adaptor molecule-1, rhodopsin, synaptophysin, postsynaptic density protein-95, retinal pigment epithelium (RPE)-specific protein-65, von Willebrand factor, a-smooth muscle actin, desmin, and Ki-67, thus enabling visualization of retinal neuronal and glial cells, photoreceptors, synapses, RPE, blood vessels, myocytes, macrophages, or cell proliferation. Using ISH, transcripts of vascular endothelial growth factor A, angiopoietin-2, and endothelial tyrosine kinase were visualized. This article describes for the first time in minipig eyes speckle noise-free OCT, AngioOCT, and a set of IHC/ISH markers on Davidson's fixed paraffin-embedded tissues and helps to establish the minipig for ocular toxicology and pharmacology studies.

Correlation of spectral domain optical coherence tomography with histology and electron microscopy in the porcine retina

“…Our TEM data demonstrated that horizontal cells in the outer portion of the INL exhibited abundant cytoplasm, eccentric nuclei, and cytoplasmic processes containing numerous mitochondria (Figure 4 and Xie et al, 2018 [Figure 5]). Using immunohistochemical and immunofluorescent techniques, we demonstrated positive labeling of the horizontal cells with anti-calbindin antibody (Figures 3A and 3B), which is consistent with previous studies showing calbindin-labeled horizontal cells in the pig (Atzpodien et al, 2016; Ghosh and Arner, 2010; Johansson et al, 2010). Our observations of the histologic and ultrastructural characteristics of the porcine horizontal cells are also consistent with a previous ultrastructural study (Beauchemin, 1974).…”

“…The pig model is being increasingly used to study retinal physiology and pathophysiology related to human diseases (Hein et al, 2012; Hein et al, 2015; Hein et al, 2016; Lim et al, 2018), and several investigators have used the porcine eye to study the application of intraoperative OCT for human vitreoretinal surgery (Asami et al, 2016; Ehlers et al, 2014; Hahn et al, 2013; Li et al, 2014). Recently, investigators have employed advanced clinical imaging techniques, including swept source and spectral domain OCT, and tissue-based biomarkers in the porcine eye in an attempt to reduce the utilization of nonhuman primates for toxicology studies (Atzpodien et al, 2016); however, the correlation between OCT images and the corresponding lamellar architecture and cellular constituents of the retina was not addressed or documented even though the anatomical attribution of OCT signals in the human retina have been studied extensively (Cuenca et al, 2018; Spaide and Curcio, 2011; Staurenghi et al, 2014). With advancement in OCT technology, the number and definition of the different bands on OCT images of the outer retina has been modified significantly in the last two decades (Jonnal et al, 2014; Spaide, 2012).…”

The Minipig is a Suitable Non-Rodent Model in the Safety Assessment of Single Stranded Oligonucleotides

“…PCSK9- or APOB-specific mRNA target probe sets ( N   =  20) for Sus scrofa were provided by Exiqon (gene ID 100620501, targeted bps 2 through 1464 of the PCSK9 cDNA sequence; Gene ID 100523371, targeted bps 3564 through 4553 of the APOB cDNA sequence). Following signal amplification, sections were stained with Fast Red and counterstained with hematoxylin as previously described (Atzpodien et al, 2016). All steps of this procedure were carried out using a Ventana Discovery Ultra ® immunostainer.…”

FluoroGold-Labeled Organotypic Retinal Explant Culture for Neurotoxicity Screening Studies

“…For this purpose, rabbit, guinea pig, or rat models are the most commonly utilized as a basis for preclinical studies. In the case of bigger animals, that is, rabbits, ophthalmic examination, including funduscopy, fluorescein angiography, or optical coherence tomography can be performed [ 7 – 9 ]. However, there are growing evidences of applicability of optical coherence tomography in retina studies involving also small rodents [ 10 – 12 ].…”